• Korean Journal of Microbiology
• /
• v.43 no.3
• /
• pp.159-165
• /
• 2007

For the development of basic genetic materials for specific and effective therapeutic approach to suppress multiplication of hepatitis C virus (HCV), HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was developed. The ribozyme targeted most effectively to +382 nucleotide (nt) site of HCV IRES RNA. The allosteric ribozyme was designed to be composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nt of HCV IRES. Noticeably, we employed in vitro selection technology to identify the most appropriate communication module sequence which can induce ribozyme activity depending on the US5B protein. We demonstrated that the ribozyme was nonfunctional either in the absence of any proteins or in the presence of control bovine serum albumin. In sharp contrast, the allosteric ribozyme can induce activity of cleavage reaction with HCV IRES RNA in the presence of the HCV NS5B protein. This allosteric ribozyme can be used as lead compound for specific and effective anti-HCV agent, tool for highthroughput screening to isolate lead chemicals for HCV therapeutics, and ligand for biosensor system for HCV diagnosis.

• BMB Reports
• /
• v.54 no.2
• /
• pp.118-123
• /
• 2021

The bacterial effector protein RavZ from a pathogen can impair autophagy in the host by delipidating the mammalian autophagy-related gene 8 (mATG8)-phosphatidylethanolamine (PE) on autophagic membranes. In RavZ, the membrane-targeting (MT) domain is an essential function. However, the molecular mechanism of this domain in regulating the intracellular localization of RavZ in cells is unclear. In this study, we found that the fusion of the green fluorescent protein (GFP) to the MT domain of RavZ (GFP-MT) resulted in localization primarily to the cytosol and nucleus, whereas the GFP-fused duplicated-MT domain (GFP-2xMT) localized to Rab5- or Rab7-positive endosomes. Similarly, GFP fusion to the catalytic domain (CA) of RavZ (GFP-CA) resulted in localization primarily to the cytosol and nucleus, even in autophagy-induced cells. However, by adding the MT domain to GFP-CA (GFP-CA-MT), the cooperation of MT and CA led to localization on the Rab5-positive endosomal membranes in a wortmannin-sensitive manner under nutrient-rich conditions, and to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8-PE. Furthermore, GFP-CA△α3-MT, an α3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8-PE. Thus, the CA or MT domain alone is insufficient for stable membrane localization in cells, but the cooperation of MT and CA leads to localization to the endosomal and autophagic membranes. In autophagic membranes, the CA domain can delipidate mATG8-PE without requiring substrate recognition mediated by LC3-interacting region (LIR) motifs.

• Parasites, Hosts and Diseases
• /
• v.38 no.2
• /
• pp.107-110
• /
• 2000

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g. HSP70-NH2-terminal region, or rT.g. HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NHa-terminal region did not. These results suggest that T.g. HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.9
• /
• pp.1492-1495
• /
• 2008

Successful control of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak, requires a specific and reliable diagnostic tool. A pathovar-specific PCR assay was developed for the rapid and accurate detection ofthe plant pathogenic bacterium Xanthomonas oryzae pv. oryzicola in diseased plant. Based on differences in a membrane fusion protein gene of Xanthomonas oryzae pv. oryzicola and other microorganisms, which was generated from NCBI (http://www.ncbi.nlm.nih.gov/) and CMR (http://cmr.tigr.org/) BLAST searches, one pair of pathovar-specific primers, XOCMF/XOCMR, was synthesized. Primers XOCMF and XOCMR from a membrane fusion protein gene were used to amplity a 488-bp DNA fragment. The PCR product was only produced from 4 isolates of Xanthomonas oryzae pv. oryzicola among 37 isolates of other pathovars and species of Xanthomonas, Pectobacterium, Pseudomonas, Burkholderia, Escherichia coli, and Fusarium oxysporum f.sp. dianthi. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.11
• /
• pp.1529-1535
• /
• 2013

Tuberculosis is a worldwide epidemic disease caused by Mycobacterium tuberculosis, with an estimated one-third of the human population currently affected. Treatment of this disease with aminoglycoside antibiotics has become less effective owing to antibiotic resistance. Recent determination of the crystal structure of the M. tuberculosis Rv3168 protein suggests a structure similar to that of Enterococcus faecalis APH(3')-IIIa, and that this protein may be an aminoglycoside phosphotransferase. To determine whether Rv3168 confers antibiotic resistance against kanamycin, we performed dose-response antibiotic resistance experiments using kanamycin. Expression of the Rv3168 protein in Escherichia coli conferred antibiotic resistance against $100{\mu}M$ kanamycin, a concentration that effected cell growth arrest in the parental E. coli strain and an E. coli strain expressing the $Rv3168^{D249A}$ mutant, in which the catalytic Asp249 residue was mutated to alanine. Furthermore, we detected phosphotransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in E. faecalis APH(3')-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis, likely through phosphotransferase targeting of kanamycin.

• Proceedings of the Korean Society of Toxicology Conference
• /
• 2002.05b
• /
• pp.1-19
• /
• 2002

It is well established that the initiating event in chemical carcinogenesis is the binding of reactive carcinogens to DNA. Thus, a number of analytic methods have been developed for determining levels of carcinogen-DNA adducts in humans as a marker of individual exposure and, potentially, of risk for cancer development. In addition, reactive carcinogens also bind to protein suggesting protein adducts can be used as a surrogate for DNA adducts in some situations. We have developed monoclonal and polyclonal antibodies to carcinogen-DNA and protein adductis and highly sensitive ELISA and immunohistochemical assays for determining levels of adducts in human tissues. These studies have demonstrated higher levels of adducts in those with higher exposure as a result of workplace, dietary, chemotherapy, environmental of lifestyle (smoking) exposures. Elevated levels of adducts have been found in lung and liver cancer cases compared to controls. We have also used DNA adducts to determine efficacy of an antiosidant vitamin intervention. DNA adduct studies have demonstrated very different levels of damage in those with similar exposure levels. These interindividual differences are likely the result genetic differences in capacity to activate carcinogens, detoxify reactive intermediates and repair DNA adducts once formed. We are currently investigating the relationship between polymorphisms in a number of these genes to determine their relationship to adduct levels as well as their ability to confer increased risk for cancer development. The ability to identify high risk individuals will allow the targeting of screening and preventive strategies to those most likely to benfit.

• Molecules and Cells
• /
• v.21 no.3
• /
• pp.389-394
• /
• 2006

Human seven-in-absentia (SIAH)-interacting protein (SIP) is a component of the E3 ligase complex targeting beta-catenin for destruction. Arabidopsis has one SIP protein (AtSIP) with 32% amino acid sequence identity to SIP. To investigate the functions of AtSIP, we isolated an atsip knockout mutant, and generated transgenic plants overexpressing AtSIP. The growth rates and morphologies of the atsip and transgenic plants were indistinguishable from those of wild type. However, atsip plants were more susceptible to Pseudomonas syringae infection, and the transgenic plants overexpressing AtSIP were more resistant. Consistent with this, RNA blot analysis showed that the AtSIP gene is strongly induced by wounding and hydrogen peroxide treatment. In addition, when plants were infected with P. syringae, AtSIP was transiently induced prior to PR-1 induction. These observations show that Arabidopsis AtSIP plays a role in resistance to pathogenic infection.

• The Korean Journal of Zoology
• /
• v.35 no.2
• /
• pp.161-172
• /
• 1992

In the short-term treahent of 12-0-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF), the'Wh and PDGF induced the Protein Kinase C (PKC) activation and migration from the cytoplasm to the peripheral nulcear membrane. And the activated PKC which was directly or indirectly stimulated by TPA or PDGF Phosphorylated many kinds of PKC's targeting proteins and induces various biological responses. Especially, the cytoplasmic PKC was phosphorylated within 1 hr and 10 min by TPA-and PDGF-treahent respectivelv. In the long-term treatment of TPA or PDGF, both of them induced the down-regulation and translocation of PKC in the mvoblasts. The down-regulation of PKC isozyrnes, the pattern of PKC I and ll was similar to the PKC 111 isozpnes in the cytoplasm. But in the nucleolus, the TPA did not induce and down-regulation or the inhibition of the immunoreactivity of PKC III antibody. This investigation indicates that each isozvmes of PKC mal be performed the different effects to the down-regulation of the cytoplasm or nucleolus. And douvn-regulated myoblasts contained low immunoreactivity of PKC antibodies.

• Tuberculosis and Respiratory Diseases
• /
• v.75 no.6
• /
• pp.236-237
• /
• 2013

Many attempts have been made to find genetic abnormalities inducing carcinogenesis after the development of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor targeting EGFR in lung cancer. New target therapies have been already commercialized and studied along with the recent discovery of gene rearrangement involved in the carcinogenic process of non-small cell lung cancer. This study aims to investigate anplastic lymphoma kinase, c-ros oncogene 1, and receptor tyrosine kinase, in particular.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1998.11a
• /
• pp.192-192
• /
• 1998

The blood - brain barrier (BBB) expresses high concentrations of transferrin receptor, and it was revealed that anti-transferrin receptor mouse monoclonal antibody (OX26) undergoes transcytosis through the BBB. This property allows the OX26 to serve as a brain drug delivery vector. In an attempt to produce broadly useful targeting agents, genetic engineering and expression techniques have been used to produce antibody-avidin (AV) fusion protein (OX26 IgG3C$\_$H/3-AV). In the present study we estimated the BBB permeability and stability of genetically engineered vector.